Project Summary: Melanoma is a high-risk cancer of melanocytes ? skin cell types that make melanin. Melanoma is the most serious of the skin cancers and for which development of effective therapeutic solutions has been difficult. Chemotherapy, immunotherapy, and targeted signal transduction pathway drugs are current options for stage III and IV treatments if the tumor is metastatic, yet cytotoxicity and development of resistance are persistent concerns. The current paradigm suggests that combined therapeutic approaches will likely bring the best survival rates ? in particular, if different mechanisms of action can be targeted. Palmerolides are bioactive macrolide natural products isolated from the Antarctic ascidian, Synoicum adareanum that offer this potential. The compounds are hypothesized to be produced by a mixed non-ribosomal peptide synthase/polyketide synthase biosynthesis gene cluster that is encoded in the genome of bacterial symbionts associated with the host ascidian. Palmerolides target melanoma with a low LC50 (18 nM) and have nM-level cytostatic activity toward several other cell lines, thus are highly specific. The mode of action appears to target transmembrane vacuolar ATPases (V- ATPase) that offer significant potential as specific drug targets. As chemotherapeutic agents, the natural source of palmerolides (Antarctic ascidian) will be insufficient to supply clinical use, chemosynthesis is not currently viable and cultivation efforts have been unsuccessful in bringing the palmerolide-producing organism into culture as of yet. Therefore, we have taken an approach that will lead to a sustainable supply of palmerolide that does not rely on harvesting or cultivation of the producing organism by going directly to the multi-species metagenome of the bacterial symbionts of S. adareanum. The approach of metagenome bioinformatic mining has re-invigorated the field of natural product discovery and has led to the identification of numerous new natural product biosynthetic gene clusters, and is the approach that is proposed here. Three specific aims are proposed that will lead to identification of the Pal producing gene cluster in the metagenome of the S. adareanum microbiome. Aim 1: Assemble, annotate and mine the S. adareanum microbial metagenome. Aim 2. Apply a predictive bioorganic synthesis strategy from organic structural and genome architecture contexts to identify potential Palmerolide biosynthetic gene clusters. Aim 3. Validation of candidate organism(s) gene content using single cell genome sequencing. The outcomes of this research will result in identification of the Pal producing organism and its biosynthetic machinery, and form the basis for downstream research required for Pal development.